From the end of the nineteen eighties it has been known that interleukin-8 (IL-8) is a potent neutrophil agonist. Among other functions IL-8 induces Ca.sup.++ ion flow into neutrophils, the increase in intracellular Ca.sup.++ concentration ([Ca.sup.++ ].sub.i) being the starting event which triggers the activation of neutrophils and of other leucocytes, causing L-selectin release, chemotaxis and subsequent degranulation in the presence of cytoclasin C.
Recurring evidence indicates the chemokine IL-8 to be implicated in maintaining, increasing and amplifying neutrophil-dependent inflammatory symptoms which characterize a great number of diseases such as psoriasis (B. J. Nicholoff et al., Am. J. Pathol. 138, 129, 1991), rheumatoid arthritis (M. Seitz et al., J. Clin. Inv. 87, 463, 1991), ulcerative colitis (V. R. Mahida, Clin. Sci. 82, 273, 1992), pulmonary idiopathic fibrosis and acute respiratory failure (P. C. Carre et al., J. Clin. Invest. 88, 1802, 1991 and E. J. Miller et al., Am. Rev. Respir. Dis. 146, 427, 1992) and also to have a decisive role in amplifying the damage due to reperfusion (N. Sekido et al., Nature 365, 654, 1993). In fact, high amounts of IL-8 were found in sputum and in oedematous fluids of patients suffering from chronic inflammatory diseases of the respiratory tract, including from cystic fibrosis up to pulmonary obstructive diseases, such as chronic bronchitis, bronchiectasia, atelectasia, which all are characterized by intrapulmonary accumulation of polymorphonucleate leukocytes (PMN) (J. B. Y. Richman-Eisenstat et al., Eur. Respir. J. 6, 1429, 1993 and H. Nakamura et al., Am. J. Respir. Crit. Care Med. 149, 1037, 1994).
The more evident characteristics of the above diseases are chronic bacterial infection as well as accumulation of high amounts of PMN neutrophils in the airways. PMN neutrophils in turn are responsible for inducing tissue damage and hypersecretion. The recurring bacterial infections which characterize the often unfavourable disease course, co-operate in increasing these symptoms. Pseudomonas aeruginosa, one of the most widespread infecting microorganisms in such diseases, is characterized by the property of inducing and stimulating the production of IL-8 from the epithelial cells of the respiratory tract, so that, contributing to neutrophil activation, it worsens tissue damage, either indirectly, through the release of neutrophil enzymes, such as elastase and catepsins, or directly, causing the formation of O.sub.2 radicals and of hypochlorous acid, i.e. of cytotoxic species (P. P. Massion et al., J. Clin. Inv. 93, 26, 1994 and P. J. Jorens et al., 263, 1708, 1993).
In experimental models of glomerulonephritis in rabbits, induced by endotoxin or bovine albumin, the intravenous administration of antibodies against IL-8 had a favourable effect as shown by marked decrease of urinary protein excretion, from 3.2 mg/h to 0.9 mg/h, and by prevention of podocyte fusion in the glomerulus (T. Wada et al., J. Exp. Med. 180, 135, 1994).
As for other cytokines, the selective inhibition of the synthesis or of the action of IL-8 may result in a therapeutical advantage; one of the possible ways to reach such a goal could be to neutralize cytokine activity in extracellular fluids and in haematic circulation by using antibodies or soluble receptors or proteins able to bind IL-8 as an alternative to the use of receptor antagonists.
4-[3-(4-Fluorophenyl)]-2-[[4-(N-3-(2-quinolinmethoxy) phenyl)]amino]phenylpropylbenzoic acid (ETH-615), a well known leukotriene synthesis inhibitor, clearly inhibited the chemotaxis of PMN leukocytes induced by the cytokine IL-8 or by the leukotriene LTB.sub.4, but only slightly inhibited that one induced by N-formyl-methionyl-leucyl-phenylalanine (FLMP or fLMP). Even if it has no effects on T-cell migration stimulated by these agonists, it has been recently proposed in clinical studies for the therapy of inflammatory diseases of the skin, characterized by high levels of leukotrienes IL-8 and LTB.sub.4 (M. Kristensen et al., Exper. Dermatol. 2, 165, 1993).
The increase of [Ca.sup.++ ].sub.i in PMN neutrophils is the event which marks and induces their activation following stimulation by different agonists, including, among endogenous stimulants, the leukotriene LTB.sub.4, PAF and the C5a factor of the complement, besides IL-8 and the synthetic tripeptide N-formyl-methionyl-leucyl-phenylalanine (FLMP). The transduction of the signal, both at the receptor and post-receptor levels, which causes an increase of [Ca.sup.++ ].sub.i in these cells, is stimulus-dependent and the production of the superoxide anion may be taken as a measure of said activation.
Leumedine or N-Fmoc-L-leucine (NPC-15669), which belongs to a Fmoc-aminoacid serie, inhibited FLMP-stimulated, but not other agonist-stimulated [Ca.sup.++ ].sub.i increase in PMN neutrophils (R. J. Smilth et al., Brit. J. Pharmacol. 114, 1694, 1995). Other leumedines have been described inhibiting IL-8-mediated accumulation of neutrophils in the respiratory tract of dogs (P. G. Jorensen et al., Europ. Respir. J. 7, 1935, 1994).
It is also known that non steroidal antiinflammatory drugs (NSAIDs), while inhibiting the synthesis of prostaglandines (PGs), do not interfere with the production and release of chemotactic chemokines MCP-1 and IL-8. On the contrary, in a comparative study, dexamethasone, in an optimal manner, and some other antirheumatic drugs, such as sodium thiomalate and metotrexate, in a sub-optimal manner, were found to inhibit the release of these cytokines thus suggesting that part of the antirheumatic activity of glucocorticoids may be due to the prevention of the accumulation of chemotactic cytokines acting on neutrophils and monocytes (P. Loetscher et al., Cytokine 6, 162, 1994).
So, for example, in human synovial cells the production of IL-8 stimulated by interleukin-1 and TNF-a, was not inhibited by the usual non steroidal antiinflammatory agents, such as thioprofenic acid, indomethacin, naproxen and piroxicam (P. Loetscher et al., Cytokine 6, 162, 1994).
Development of oedema in an inflamed site seems to need the contemporary presence of IL-8 and PGE.sub.2, while the single cofactors, administered by intradermal route, were unable to cause oedema formation even if a certain pro-oedemateous effect was described for cytokine IL-8 alone (I. Colditz, ididem 134, 755, 1989).
The use of (.+-.) ibuprofen, or p-isobutylhydratropic acid, and of (.+-.) flurbiprofen, or 3-fluoro-4-phenylhydratropic acid, as well as of their corresponding C1-8-alkyl esters and of pharmaceutically acceptable salts thereof, has been described and claimed for the treatment of respiratory diseases, particularly in the treatment of acute respiratory failure in EP 070 714 (Jul. 5, 1986). (.+-.) Ketoprofen, (.+-.) ibuprofen and (.+-.)flurbiprofen and naproxen are examples of NSAIDs widely used in the therapy of a number of diseases. Ketoprofen, ibuprofen and flurbiprofen are used as racemates, while naproxen is utilized only in the form of the (S) enantiomer. The treated diseases include, besides tooth ache and other painful symptoms, acute inflammation, rheumatic and degenerative diseases of the joints, blood platelet adhesion and, in the case of ibuprofen, also cardiac infarction.
It is believed that, similarly to acetylsalicylic acid, the therapeutic potency and effectiveness of these 2-arylpropionic acids is due to their common property of inhibiting the cyclooxygenase enzyme (CO) which transforms arachidonic acid into algogenic, pro-inflammatory PGs, of which PGE.sub.2 is the most representative model.
PGs have an important role in the production of pain, inflammation and fever and, consequently, the above mentioned compounds are used as analgesic, antiinflammatory and antipyretic drugs.
When they are administered as a single dose or as short-term intermittent therapy, they provide suitable analgesia and can clear up pain of slight to moderate intensity, while, in the majority of cases, it is necessary administering them for several days, and also for weeks, in order to obtain a clear antiinflammatory effect.
Even if several comparative studies between single non steroidal antiinflammatory compounds or studies comparing a single compound with many others were published, an overall comparison allowing to build up an effectiveness order list is lacking. It is usually believed that only small differences in activity exist and drug choice by physicians is generally made on an empirical basis. Moreover, the individual responses of patients may greatly vary among each other, so that if a patient does not respond to a given drug, he may be treated with another one. It is however recommended to prefer the use of NSAIDs with the lowest risk of gastroenteric toxicity and at the minimum active dose. More recently it has been supposed that NSAIDs act through the inhibition of two isoforms of the cyclooxygenase (COX-1 and COX-2); the inhibition of COX-1 would be associated with the gastroenteric side effects which are sometimes observed in the treatment with the arylacetic and 2-arylpropionic acids, while those NSAIDs which are highly selective against COX-2 would possess less gastroenteric toxicity (Martindale, The Extra Pharmacopoeia, 31st Ed., 72, 1996).
The enzymatic inhibition process of the two isoforms of Co, i.e. COX-1 and COX-2, and consequently the block of the pro-inflammatory, pro-algogenic and pro-pyretic PGs, is a stereospecific process.
Only the (S) enantiomers of the 2-arylpropionic acids, which inhibit PGE.sub.2 production, are considered effective as antinflammatory agents (D. Mauleon et al., Drugs 52, 24, 1996).
The (R) enantiomers have practically no effect on the enzyme and on PG synthesis; some activity is observed only at very high concentrations, from 100 to 1000 times greater than those of the other enantiomer, and higher than blood levels obtained after administration of these substances (10.sup.-9 -10.sup.-6 M). By consequence the (R) enantiomers of 2-arylpropionic acids were considered for a long period of time as being devoid of any interesting therapeutic utility.
Actually, they are converted in vivo, particularly in the liver and only in negligeable amounts in other tissues such as mouse peritoneal macrofages, into the (S) enantiomers through stereoselective activation of their thio-esters by means of CoA (S. Menzel-Soglowek et al., Biochem. Pharmacol. 43, 1487, 1992) and thus they contribute to the global activity of the racemate. The extent to which such bio-conversion takes place in vivo is dependent upon animal species and chemical structure of the compound. So, for instance, the (R) enantiomers of ibuprofen are nearly completely converted into the (S) enantiomers in men and rats, whereas the (R) enantiomers of flurbiprofen and of ketoprofen are practically not converted (&lt;5%) in men and guinea-pigs, but completely converted in rats (K. Brune et al., Experientia 47, 257, 1991; K. Brune et al., J. Clin. Pharmacol. 32, 944, 1992).
Rats have always been the preferred species for the usual experimental models of inflammation, algesia and hyperalgesia. However, based on the recent studies showing high rate of enantiomeric conversion of 2-arylpropionic acids in this species, it seems not very suitable, since it does not allow to predict the real activity of these compounds in men, where the conversion may not take place or occur only to a very low extent. In fact, only recently the (R) enantiomers of such 2-arylpropionic acids which, like flurbiprofen and ketoprofen, are not metabolically activated in men, were demonstrated to inhibit pain perception in men with an efficacy at least similar to that of the (S) enantiomers (K. Brune et al., Experientia, 1991).
In order to predict the therapeutic effect of the racemates in men, it is necessary to know how much the single enantiomers contribute to the global activity, using experimental models excluding metabolic bio-conversion. This is possible utilizing guinea-pigs instead of rats as experimental animal species in the classical experimental model of subplantar carrageenin injection into the right paw (P. Ghezzi et al., J. Pharmacol. Exp. Ther., 1997). This model allows the contemporary evaluation of the inhibition of oedema formation and of hyperalgesia.
The L-lysine salts of (S)- and (R)-ketoprofen were evaluated by means of the aforesaid test in guinea-pigs in comparison with the L-lysine salt of ketoprofen racemate using indomethacin, an achiral arylacetic acid, as positive internal standard. In the dose range from 25 to 750 mmoles/kg, the salt of the (S) enantiomer inhibited oedema formation in a dose-dependent manner, reaching a statistically significant effect at 75 mmoles/kg and the maximal effect at 250 mmoles/kg and showing less effect at higher doses. Also the salt of the (R) enantiomer significantly inhibited oedema formation, but only starting from the 250 mmoles/kg dose; the activity was dose-dependent also in this case, although with a different slope of the dose-effect curve, thus indicating a different mechanism of action. On the contrary, the L-lysine salt of (R)-ketoprofen showed a marked, dose-dependent inhibitory effect on hyperalgesia at doses from 75 to 250 mmoles/kg. This effect was maximal at the highest dose. The L-lysine salt of (S)-ketoprofen displayed only a slight inhibitory effect on hyperalgesia, wich was statistically significant only at the highest dose of 750 mmoles/kg. The anti-oedema and anti-hyperalgesic effects of the L-lysine salt of the racemate were constantly intermediate between those evaluated for the two enantiomers taken alone, thus indicating that, in the absence of any bio-conversion of the (R) enantiomer, the global activity of the racemate is to be attributed to the (S) enantiomer as far as the anti-oedema activity is concerned and to the (R) enantiomer in regard to hyperalgesia inhibition. This conclusion is substantially according to what reported in the aforesaid paper by K. Brune.
Guinea-pigs are a species naturally resistant to the gastrolesive action of NSAIDs, so that it is not possible to compare the two enantiomers in respect to this parameter in this species, unless using very high doses, having no predictive value. To this purpose, rats are necessarily, again, the chosen species, although the least suitable. The results of a comparative study of the L-lysine salts of (S)- ant (R)-ketoprofen using this animal model clearly showed that the (R) enantiomer has less ulcer-inducing properties. The differences among the enantiomers and the racemate were statistically significant starting from the dose of 40 mmoles/kg: at this dose an "ulcer score" of 2 was evaluated for the (R) enantiomer, compared with "ulcer scores" of 3 and 4 for the racemate and the (S) enantiomer, respectively.
A parallel evaluation of the inhibitory effects of (R)- and (S)-ketoprofen L-lysine salts and of racemic ketoprofen L-lysine salt on lipopolysaccharide-stimulated PGE.sub.2, TNF-a and IL-1b release from mouse peritoneal macrophages allowed an interesting interpretation of the above disclosed matter. The L-lysine salt of (S)-ketoprofen inhibited PGE.sub.2 formation within the whole dose range of 10.sup.-6 -10.sup.-9 M, while the same effect was observed for (R)-ketoprofen only in the range 10.sup.-6 -10.sup.-5 M. On the other hand, surprisingly, the L-lysine salts of (S)-ketoprofen and of the racemate stimulated TNF-a and IL-1b formation in a dose-dependent manner, reaching the statistical significance in the range 10.sup.-8 -10.sup.-6 M in the case of TNF-a, and at the concentration of 10.sup.-5 M in the case of IL-1b. On the contrary the L-lysine salt of (R)-ketoprofen was completely ineffective and did not stimulate the release of these cytokines within the overall 10.sup.-9 -10.sup.-5 M concentration range. Speculatively, the low gastric tolerability of the lysine salts of (S)-ketoprofen and of racemic ketoprofen could be the direct consequence of the stimulation of TNF-a release ("up regulation") (C. B. Appleyard et al., Am. J. Physiol., 270, G-42, 1996) rather than of the blockade of PGE.sub.2 synthesis, as it was thought till now. Furthermore, TNF-a "up-regulation" may provide an understanding key of the lower effectiveness of the racemate salt and of the (S)-ketoprofen salt, in comparison with the (R) enantiomer salt, in the control of hyperalgesia, just for the fact that this last enantiomer, differently from the previous ones, does not amplify the formation of the inflammatory cytokines.
These results substantially agree with those obtained in a comparative study on the topic antiinflammatory activity of racemic ketoprofen and of the single enantiomers in the ultra-violet radiation-induced epidermal erythema test in guinea pigs. Protection obtained by the use of the (R) enantiomer was calculated to be 53.1.+-.4.6%, quite similar and of statistically significant (p&lt;0.05) in respect to that obtained with racemic ketoprofen (56.1.+-.3.1), and lower than that obtained with the (S) enantiomer (73.4.+-.4.0%). However, the whole results obtained in other experimental models of inflammation, such as carrageenin-induced oedema in rats and croton oil-induced ear oedema in mice, let the authors to conclude that the in vivo antiinflammatory effectiveness of the (R) enantiomer was significantly lower than those of the racemate and of the (S) enantiomer.
Similar conclusions were drawn by Svesc et al. (Chirality, 5, 589, 1993) on the basis of TBX.sub.2 synthesis inhibition in human PMN leukocytes and in rat platelets. In this test (R)-ketoprofen was active at doses 2-3 times greater than the (S) enantiomer and the racemate. On the contrary, in an acetic acid-induced inflammation model in rats, interleukin IL-8 production was significantly (p&lt;0.1) reduced from 53.8 pg/ml to 22.4 and 16.9 pg/ml after administration of (.+-.)ketoprofen 200 and 100 mg/kg, respectively (L. M. Wang et al., Drugs Exper. Clin. Res., 23, 1, 1997).
In a study on patients with initial rheumatoid arthritis the administration of 200 mg of ketoprofen for 10 days caused the normalization of the increased chemotactic index and adhesiveness and a reduction of PMN leucocyte fagocytosis, while their bactericidal function was not affected. Moreover, the chemotactic activity induced by zymosan, an activator of the complement, was inhibited both in healthy volunteers as well as in the studied patients (E. Bacino et al., Clin. Exper. Reumatol., 5, 50, 1987).
Successively, the question of a PG-synthesis inhibition-independent antiinflammatory activity of NSAIDs was widely discussed and studied, particularly in relation to the inhibition of human PMN neutrophil activity and to the mechanisms regulating the function of these cells, which are still substantially unknown. So, for example, it was thought that drugs like ketoprofen, flurbiprofen, sudoxicam, fenofren and indomethacin, after the oral administration, inhibited carrageenin-induced formation of pleural exudate in rats due to their ability to inhibit the migration of PMN cells, but not that of monocytes, into the pleural cavity (A. Blackam and R. T. Owen, J. Pharm. Pharmacol., 27, 201, 1975). But, later, it was demonstrated exatly the opposite for some of these drugs, among which ketoprofen (S. C. R. Meacock and E. Ann Kitchen, Future Trends Inflammation, Proc. Int. Meet., 2nd(1975)320, J. P. Giroud et al, Eds, Birkhaeuser, Basel).
More recently it was supposed that in the case of the fenamates, a particular subgroup of NSAIDs, such as flufenamic and tolfenamic acids, the inhibition of neutrophil activation induced by Ca.sup.++ ionophore and by the chemotactic peptide FLMP (N-formyl-methionyl-leucyl-phenylalanine) was due to the blockade of Ca.sup.++ ion entry as indicated by the results of trials evaluating Mn.sup.++ and .sup.45 Ca.sup.++ ion flow. Fenamates seem special in this respect, as compared to other NSAIDs, since ketoprofen, which is taken as the typical prostanoid synthesis inhibitor, was completely inactive like nifedipine, an inhibitor of the voltage-independent Ca.sup.++ channels, and unlike 1-[2-(4-metoxyphenyl-2-[3-(4-metoxyphenyl) propoxy]ethyl]1H-imidazole (SK&F 96365), a non selective Ca.sup.++ channel blocker (H. Kankaanranta and E. Moilanen, Molec. Pharmacol., 47, 1006, 1995).